Light and electron microscopic abnormalities in placentae from ovine somatic cell clones at term

Cloning represents a powerful tool for production of transgenic animals, study of gene functionand for the isolation of totipotent stem cells for tissue and ce11 therapy. The biggest challengefacing this technology is overcoming the high rate of pregnancy failure and fetal mortality,which seems in most cases related to a compromised placenta (Palmieri et al., Placenta 28:577-84, 2007, doi 10.1016 /j.placenta.2006.08.003).T o investigate the reasons for fetal losses afierSCNT, an immunohistochemical (MC) and ultrastnictural analysis of 3 ovine cloned placentaeat term (147 days) was performed; 5 control placentae obtained afier natura1 mating also wereevaluated.Fetal cotyledons were fixed in buffered 10% formalin for histology (H & E, PAS) and IHC(smooth muscle actin, desmin, calponin, CD34, and vWF). Small pieces of the same sampleswere fixed in 2.5% gluteraldehyde, post-fixed in 1% Oso4 and embedded in epoxy resin forTEM investigation. Irnage analysis was performed on semithin sections (1 pm) (fetal capillaries:diameter, cross-sectional area, no.capillaries/mm2, capillary total arealvillous area) and onultrathin sections (75 nm) (subtrophoblastic basement membrane-SBM-thickness usingorthogonal intercepts method).We observed a marked reduction of villous vascularisation (503 capillaries/mm2 in control and50.7/mm2 in cloned), hypoplasia of trophoblastic epithelium, lack of binucleate cells, immaturityof placenta1 vessels (reduced expression of vWF and calponin) and reduced vasculogenesis(lacked of CD34 immunoreactivity). In clones, a dimise thickening (cloned: 358 * 0.28 nrn;control: 132 * 0.94 nm) and lamination of SBM and capillaries basa1 lamina were noted.We hypothesize that abnormal vascularisation, ischaemia and low development of specializedtrophoblastic epithelium were the primary causes of fetal losses after SCNT. The main focus onthe placenta as the organ limiting norma1 development of clones suggests severa1 strategies toovercome this hurdle and provide additional data in the light of intensive research efforts toimprove potential applications of SCNT.[...]